Many laboratory and clinical procedures employ bio-specific affinity reactions. Such reactions are commonly utilized in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cells, viruses, proteins, nucleic acids and the like. Various methods are available for analyzing or separating the above-mentioned target substances based upon complex formation between the substance of interest and another substance to which the target specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, Whitehead et al. (U.S. Pat. No. 4,554,088) and Hunter et al. (Immunoassays for Clinical Chemistry, pp. 147-162, eds., Churchill Livingston, Edinborough, 1983). Generally, any material that facilitates magnetic or gravitational separation may be employed for this purpose. More recently, the superiority of magnetics for performing such separations has led to its use in many applications.
A problem with magnetic separation protocols is that magnetic beads must be added in excess to a sample to ensure a sufficient amount of binding of beads to a target analyte in the sample, thus producing a sample that contains a very high percent of magnetic particles that are not bound to target analytes, as well as non-specific target entities. Non-specific target entities may for example be bound at a much lower efficiency, for example 1% of the surface area, while a target of interest might be loaded at 50% or nearly 100% of the available surface area or available antigenic cites. However, even 1% loading may be sufficient to impart force necessary for trapping in a magnetic gradient flow cell or sample chamber.
The presence of magnetic particles that are not bound to target analytes and non-specific target entities on the surface that includes the target/magnetic particle complexes interferes with the ability to successfully separate the target of interest of the remaining components of the mixture and unbound magnetic particles. The magnetic capture of the resulting mix, and close contact of magnetic particles with each other and labeled targets, result in the formation of aggregate that is hard to dispense and which might be resistant or inadequate for subsequent processing or analysis steps.
There is a need for methods for separating target analytes from a sample.